Mast cells are multifunctional immune cells that play an important role in host defense but also promote asthma, allergic rhinitis, rheumatoid arthritis, atherosclerosis, skin-blistering diseases, cancer metastasis and are implicated in periodontal disease. There are important phenotypic and functional differences between human and rodent mast cells. Not surprisingly, therapeutic approaches developed using rodent models for mast cell-mediated disease such as asthma lack efficacy in humans. Thus, to better understand the mechanisms of mast cell-mediated diseases, it is critical to utilize in vivo models that are relevant to humans. In this proposal, the major objective is to develop a humanized mouse model for the genetic manipulation of human mast cell function in vivo. Aggregation of high affinity IgE receptors (FcRI) on mast cells leads to the generation of C3a, which causes substantial degranulation in human mast cells and likely contributes to IgE-mediated allergic responses in vivo. Recent studies performed in our laboratory have identified novel signaling pathways via which G protein coupled receptor kinases (GRK5, GRK6) and adapter molecules -arrestin 1 and Na+/H+ exchange regulatory factors (NHERF1 and NHEFR2) contribute to C3a-induced mast cell degranulation. We have also shown that GRK2 and GRK3 desensitize C3a-induced responses via the agonist-induced phosphorylation of C3aR. Given that C3a likely contributes to IgE-mediated responses in vivo, we hypothesize that modulation of C3aR signaling in mast cells regulates allergic responses ex vivo and in vivo. In aim #1, we will optimize conditions for the differentiation of human CD34+ cells into functional human mast cells in two strains of immune-deficient mice expressing growth factors for human mast cells. We will then target one protein that contributes to (NHERF1) and one that inhibits (GRK3) C3a-induced mast cell degranulation. Lentiviral shRNA will be used to silence the expression of these proteins in human cord blood- derived CD34+ cells and engrafted into immune deficient mice for the development of genetically manipulated human mast cells in vivo. We will use this novel approach as a proof of concept study to test the idea that genetic manipulation of human mast cells in vivo can be used to modulate bronchoconstriction ex vivo. C3aR expressed in mouse mast cells contribute to experimental passive cutaneous anaphylaxis (PCA) but not passive systemic anaphylaxis (PSA). In aim #2, we will utilize humanized mice to test the hypothesis that the presence of C3a-responsive human mast cells in the lung contributes to increased vascular pulmonary permeability in PSA. We will engraft NHERF1 or GRK3-silenced human CD34+ cells into immune deficient mice and test their impact on PCA and PSA reactions in vivo. We believe that genetic manipulation of human mast cells in humanized mice will provide a better understanding of their role in allergic and non-allergic diseases and may eventually lead to the development of novel treatment options.